[Histonet] problem in dual staining of CD4 and FOXP3

Thu Nov 8 11:59:10 CST 2007

Hi, Mark,

  Yes, I am looking at T-regulatory cells of mouse. For CD4, I 
used AF594(red color), for FOXP3 I used AF488(green color). The 
problem for me is for the second primary antibody(or maybe I 
should say for the secondary color) of dual staining, it never 
worked. I do not know which serum I should use to block the 
non-specific staining of the secondary antibody. If you think my 
protocol is imcomplete, could give me some suggestions? Many 
thanks,

Ann

On Thu Nov 08 12:45:24 EST 2007, "Tarango, Mark" 
<mtarango <@t> nvcancer.org> wrote:

> Looking at T-regulatory cells, huh?  Are you trying to stain both 
> in the
> same color?  I know Foxp3 is nuclear and CD4 is on the cell 
> membrane,
> but two colors might be better.  Your protocol looks incomplete.  
> Mark Adam Tarango HT(ASCP)
> Histology & IHC Supervisor
> Nevada Cancer Institute
> One Breakthrough Way
> Las Vegas, NV  89135
> Direct Line (702) 822-5112
> Treo (702) 759-9229
> Fax (702) 939-7663
>   -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of
> FU,DONGTAO
> Sent: Thursday, November 08, 2007 9:13 AM
> To: Histonet <@t> lists.utsouthwestern.edu
> Subject: Re: [Histonet] problem in dual staining of CD4 and FOXP3
> 
> Hello,
> 
>   Can anyone give me some suggestions on my case? I know a lot of 
> people here have a lot of dual staining experiences. I did dual 
> staining on 2 antibodies from different species in the past. They 
> worked well. But using 2 antibodies from same species is my first 
> time try and I met a big problem. Any suggestions I will be very 
> appreciate.
> 
> Ann
> 
> 
> On Thu Nov 08 08:38:39 EST 2007, "FU,DONGTAO" <fudo <@t> ufl.edu> 
> wrote:
> 
>> Hi, all
>> 
>>   Thank you first for giving me some good suggestions on 
>> thick-section question I posted last time. Now I met another 
>> problem when I did CD4 and FOXP3 dual staining using murine 
>> fresh frozen spleen. If I did single staining, both antibodies 
>> worked very well. However if I did dual staining, I could only 
>> get good result from the first antibody. The second one did not 
>> work at all(I mean no specific staining). I used CD4 from BD(rat 
>> anti-mouse). FOXP3 I used from ebioscience, also rat anti-mouse. 
>> I think there might be something wrong with my serum block(I 
>> used normal rat serum block) before I added secondary antibody. 
>> Or any other serum block I need to add to decrease the 
>> non-specific binding which I have not done yet. Does anyone can 
>> give me some suggestions according to my protocol below? How can 
>> I get specific staining of the secondary (primary)antibody?  
>> Thank you,
>> 
>>   Below is the simple protocol I used for dual staining:
>>   1. 2% normal goat serum block 20 min
>>   2. 1* antibody CD4 1:750 in diluent O/N 4C
>>   3. Seconday AF594 GT anti-rat 1:1k in 1xTBS 1h RT
>>   4. Serum block:  5% normal rat serum 30 min
>>   5. 1* antibody FOXP3 1:100 in diluent 1h RT
>>   6. Secondary AF488 Donkey anti-rat in TBS 1h RT
>> 
>>   Use 1xTBS as wash buffer. Before staining, fix tissue in -20C 
>> Aceton for 5 min, then airdry.
>> 
>> 
>> Ann Dongtao Fu MD Ph.D
>> Lab Manager
>> Molecular Pathology core
>> Dept. of Pathology
>> University of Florida
>> 1600 SW Archer Road
>> Gainesville, FL 32610
>> Lab Phone: 352-273-7752
>> FAX: 352-273-7753
>> Rm: D11-50
>> 
>> 
>> _______________________________________________
>> Histonet mailing list
>> Histonet <@t> lists.utsouthwestern.edu
>> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>> 
>> 
> 
> 
> 
> Ann Dongtao Fu MD, Ph.D
> Lab Manager
> Dept. of Pathology
> Lab phone: 352-273-7752
> Lab FAX: 352-273-7755
> Lab address: D11-50
> PO Box: 100275
> 1600 SW Archer Road
> University of Flodrida
> Gainesville, FL 32610
> 
> 
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> 
> "EMF <nvcancer.org>" made the following annotations.
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Ann Dongtao Fu MD, Ph.D
Lab Manager
Dept. of Pathology
Lab phone: 352-273-7752
Lab FAX: 352-273-7755
Lab address: D11-50
PO Box: 100275
1600 SW Archer Road
University of Flodrida
Gainesville, FL 32610